Day 1 Wet lab 22/06/15

On our first day in the lab, we began by autoclaving equipment ready for use throughout our project. Specifically, we made LB plates, LB broth and autoclaved pipette tips

Day 2 Wet lab 23/06/15

We set up overnight cultures of Top10, VS45 and MS348 cells. We then met with our supervisors to discuss our progress.

Day 3 Wet lab 24/06/15

First, we made and filter sterilised MES buffer. Then we added our Top10 cells to 250ml of LB broth containing no antibiotics. The culture was then incubated until the OD₆₀₀ was 0.6. Finally, we miniprepped pSBIC3 out of MS348.

Day 4 Wet lab 25/06/15

We set up an overnight digest of pSBIC3 ready to be run on an agarose gel in order to check that we had obtained the correct plasmid from the cells. We then transformed pSBIC3 into our competent Top10 cells. Next, we plated the transformations out onto Chloramphenicol plates and incubated them at 37ºC overnight.

Day 5 Wet lab 26/06/15

We checked the plates from overnight and calculated the competent cell efficiency. We then ran an agarose gel of the overnight digest. We decided on the layout for the wiki.

Day 6 Wet lab 29/06/15

On this day, we transformed linear pSB1C3 into our Top10 cells. We also set up overnight liquid cultures of MS349 cells containing pSB1A3. We set up a large quantity digest of pSBIC3, ready for gel extraction the following day.

Day 7 Wet lab 30/06/15

We miniprepped pSBIA3 and set up an overnight digest of it. We then ran the overnight digest of a large quantity of pSBIC3 on a gel. Finally, we had a meeting with our supervisors to discuss our progress.

Day 8 Wet lab 01/07/15

We ran a gel of purified pSBIC3 ready to be extracted, we then set up a digest of our entire stock of pSBIC3. We also transformed pSB1A3 into competent VS45 cells and plated it onto mixed Chloramphenicol and Ampicillin plates. We set up overnight cultures of MS340 containing pSBIC3.

Day 9 Wet lab 02/07/15

On this day we miniprepped pSBIC3 out of MS340. We then ran an agarose gel of the "big digest" and carried out a gel extraction procedure. We then quantified the digested material.

Day 10 Wet lab 03/07/15

We calculated the transformation efficiency of pSBIA3 in VS45 cells.

Day 11 Wet lab 06/07/15

We set up overnight cultures of Top10 cells containing pSB1A3 with limonene synthase on AMP plates, and colonies of VS45 containing pSB1A3 were patched onto chloramphenicol plates. We set up an overnight digest of miniprepped pSBIC3 using ECORI and PSTl.

Day 12 Wet lab 07/07/15

We ran an agarose gel of overnight digest, however, no bands were visible. We then set up overnight cultures of pSBIC3 and pSBIA3 to be miniprepped the next day. Finally, we set up an overnight digest of pSBIC3.

Day 13 Wet lab 08/07/15

An agarose gel of the overnight digest was run, however, no bands were visible. We then did a miniprep of pSBIA3 and pSBIC3, followed by an overnight digest of these plasmids.

Day 14 Wet lab 09/07/15

We ran another agarose gel of digested pSBIA3 and pSBIC3 - no bands were visible. We then set up overnight cultures of MS349 containing pSBIA3 LIMS and MS340 containing pSBIC3, ready to be miniprepped.

Day 15 Wet lab 10/07/15

We miniprepped pSBIA3 and pSBIC3, then focused on dry lab work for the remainder of the day.

Day 16 Wet lab 13/07/15

Overnight cultures of MS349 containing pSBIA3 LIMS and MS340 containing pSBIC3 were set up, ready to be miniprepped. We then set up an overnight digest of miniprepped plasmids pSBIA3 and pSBIC3 using the enzymes ECORI and PSTI.

Day 17 Wet lab 14/07/15

Another agarose gel of digested plasmids pSBIA3 and pSBIC3 was set up, this time it worked. We then miniprepped the overnight cultures from the night before. We then ran a gel and carried out gel extraction of both plasmids. Following this, we ran an analytical gel using Hyperladder I in order to quantify the plasmids. Finally, we set up overnight cultures of Top10 cells containing the plasmid pVS72.

Day 18 Wet lab 15/07/15

We miniprepped pVS72, then transformed it into VS45. Following the transformation, we plated the cells onto combined Chloramphenicol and Ampicillin plates. We then ran a gel of leftover digested pSBIA3 and pSBIC3 from 14/07/15. From this we were able to gel extract the plasmids.

Day 19 Wet lab 16/07/15

First, we counted the overnight colonies of VS45 with pVS72. From this, we were able to calculate the transformation efficiency.

Day 20 17/07/15

No wet lab was carried out on this day as we focused on dry lab tasks.

Day 21 Wet lab 20/07/15

We produced fresh LB agar plates containing Chloramphenicol and Ampicillin. Next, our transformed VS45 with pVS72 colonies were streaked onto fresh Chloramphenicol/Ampicillin plates and incubated overnight.

Day 22 Wet lab 21/07/15

We made Congo red plates with 0.2% L-Arabinose and 500ml LB broth containing 0.2% L-Arabinose. We then set up overnight cultures of VS45 with pVS72 in LB broth with 0.2% L-Arabinose.

Day 23 Wet lab 22/07/15

VS45 with pVS72 was plated on the Congo Red plates. We then prepared our cultures for TEM and AFM.

Day 24 Wet lab 23/07/15

We did TEM imaging, however, our results were inconclusive.

Day 25 24/07/15

We used this day to focus on dry lab tasks.

Day 26 27/07/15

We transformed Top10 cells with pVS105 (negative control plasmid) and streaked it onto Ampicillin LB plates. We also transformed VS45 with pVS72 and plated it onto Chloramphenicol/Ampicillin LB plates.

Day 27 28/07/15

We made new Chloramphenicol broth and Ampicillin broth. We then used the Amp broth to resuspend Top10 cells with pVS105. We then resuspended VS45 with pVS72 in combined Chloramphenicol and Ampicillin broth. We incubated these cultures overnight at 37ºC. For the remainder of the day, we focused on dry lab tasks.

Day 28 29/07/15

In the morning, we miniprepped the Top 10 cells containing pVS105. This will be our negative control plasmid. Following this, we diluted our culture of VS45 with pVS72 to an OD₆₀₀ of 0.1 in 3ml of LB. Once the correct OD₆₀₀ had been achieved, we spot plated 5μl of the culture onto inducing plates (containing Arabinose and IPTG), non-inducing plates and Congo red plates (both inducing and non-inducing).

Day 29 30/07/15

We transformed VS45 competent cells with the negative control plasmid (pVS105). We then plated it onto combined Chloramphenicol and Ampicillin plates.

Day 30 31/07/15

Weekend cultures of the negative control plasmid in VS45 were set up. Whilst half of the team were working in the lab, the other half attended the London meetup at London Birkbeck.

Day 31 03/08/15

We focused on dry lab tasks such as planning the questions to ask MP's and developing the questionnaire.

Day 32 04/08/15

Our transformed pVS105 in VS45 weekend cultures were spotted onto Chl + Amp plates (both inducing and non-inducing) and Congo Red. These plates were incubated overnight at 37ºC.

Day 33 05/08/15

We checked our overnight plates were checked for contamination. Following this, we transformed pVS105 and pVS72 into VS45, plated them out and then incubated them at 37ºC overnight.

Day 34 06/08/15

We set up overnight cultures of pVS105 in VS45 and pVS72 in VS45.

Day 35 07/08/15

As the colonies did not grow overnight, we set up more colonies in the morning, with the OD₆₀₀ being checked in the evening. The transformation of pVS105 and pVS72 into VS45 was then repeated. The transformations were then plated out and incubated at 37ºC.

Day 36 10/08/15

Our miniprepped plasmids (pSBIC3, pSBIA3, pVS105 and pVS72) were digested and run on an agarose gel. VS45 colonies containing pVS105 and pVS72 were resuspended in liquid LB and cultured overnight at 37ºC.

Day 37 11/08/15

First we checked the OD of the overnight cultures. We transformed pSBIA3 and pSBIC3 into Top10 cells. We made a glycerol stock solution of pVS105 and pVS72 to store it for future use. We spot plated the pVS72 and pVS105 separately onto plates for imaging. We then miniprepped pVS72 and pVS105 from the overnight cultures.

Day 38 12/08/15

First, we re-transformed pSBIA3 into Top10 cells. We then did PCR of pSBIC3 and pVS72.

Day 39 13/08/15

As PCR did not work properly the day before, we repeated the procedure. We also scanned the streaked Congo Red plates.

Day 40 14/08/15

We purified the PCR product, then digested it. We then measured the concentrations of our stock of pSBIA3 and pSBIC3.

Day 41 17/08/15

We ligated pSBIC3 and pVS72, then transformed it into competent Top10 cells. We then plated it onto Chloramphenicol plates. We used an agarose gel to test if the digest from the day before had worked successfully and also to find out if the PCR had worked. We also prepared our samples for imaging.

Day 42 18/08/15

We set up overnight cultures of the transformed ligations.

Day 43 19/08/15

We miniprepped the overnight cultures of the ligated pSBIC3 and pVS72 that were cultured overnight. Following the miniprep, we digested the plasmids. Simultaneous to the digest, we transformed 1µl of the plasmid into VS45 competent cells.

Day 44 20/08/15

We ran an agarose gel of digested plasmids from the night before. Overnight cultures of ligations of pSBIC3 and pVS72 were also set up.

Day 45 21/08/15

We miniprepped the overnight cultures. Following the miniprep, we ligated pSBIC3 and pVS72 as the previous ligations did not work. Alongside the ligations, some of the team members used Gibson assembly to fuse fragments containing CsgAss and Sup35NM to pSBIC3. The Gibson assembly products were then transformed into competent cells and incubated at 22ºC over the weekend.

Day 46 24/08/15

We carried out Gibson assembly of fragments containing CsgAss, Sup35NM and Cytochrome b₅₆₂, followed by transformation into competent cells. The ligations from the day before were digested and run on a gel. We set up overnight cultures of the Gibson assembly product from 21/08/15.

Day 47 25/08/15

We carried out PCR of fragments and set up overnight cultures of the Gibson assembled fragments that grew overnight. We then miniprepped and set up a digest of the Gibson Assembled fragments containing CsgAss and Sup35NM from the overnight cultures. From the digest, an agarose gel was run.

Day 48 26/08/15

Day 49 27/08/15

Day 50 28/08/15

Day 51 01/09/15

Day 52 02/09/15

Day 53 03/09/15

Day 54 04/09/15

Day 55 05/09/15

Day 56 07/09/15

Day 57 08/09/15

Day 58 09/09/15

Day 61 14/09/15